Method for purification of recombinant proteins

ABSTRACT

Purification of poly-amino acid-tagged recombinant proteins has been improved by the use of a carboxymethylated aspartate ligand complexed with a third-block transition metal having an oxidation state of 2+ and a coordination number of 6. A method for synthesizing the metal ion-CM-Asp complex is also described. Further, the metal ion-CM-Asp complex can be used for screening protein function.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation-in-part of co-pending applicationSer. No. 08/698,747, filed Aug. 16, 1996.

BACKGROUND OF THE INVENTION

Immobilized metal ion affinity chromatography (IMAC) was firstintroduced by Porath (Porath, J., J. Carlsson, I. Olsson, G. Belfrage[1975] Nature 258:598-599.) under the term metal chelate chromatographyand has been previously reviewed in several articles (Porath, J. [1992]Protein Purification and Expression 3:263-281; and articles citedtherein). The IMAC purification process is based on the employment of achelating matrix loaded with soft metal ions such as Cu²⁺ and Ni²⁺.Electron-donating groups on the surface of proteins, especially theimidazole side chain of histidine, can bind to the non-coordinated sitesof the loaded metal. The interaction between the electron donor groupwith the metal can be made reversible by lowering the pH or bydisplacement with imidazole. Thus, a protein possessingelectron-donating groups such as histidine can be purified by reversiblemetal complex/protein interactions.

Several different metal chelating ligands have been employed in IMAC topurify proteins. Iminodiacetic acid (IDA) ligand is a tridentate andthus anchors the metal with only three coordination sites (Porath, J.,B. Olin [1983] Biochemistry 22:1621-1630). Because of the weak anchoringof the metal, metal leakage has been known to occur. Thetris(carboxymethyl)ethylenediamine (TED) ligand is pentadentate andforms a very strong metal-chelator complex. The disadvantage of this isthat proteins are bound very weakly since only one valence is left forprotein interaction. Nitrilo triacetic acid (NTA) is a tetradentateligand which attempts to balance the metal anchoring strength withmetal-ion protein interaction properties (Hochuli, E., H. Dobeli, A.Schacher [1987] J. Chromatography 411:177-184). Other chelating ligandshave been reported and are mentioned. See, e.g., Porath (1992), supra.However, these ligands also have certain disadvantages, includingdecreased bonding capacity, decreased specificity, and increased metalleakage.

In 1991, Ford et al. (Ford, C., I. Suominen, C. Glatz [1991] ProteinExpression and Purification 2:95-107) described protein purificationusing IMAC technology (Ni-NTA ligand) as applied to recombinant proteinshaving tails with histidine residues (polyhistidine recombinantproteins). This method takes advantage of the fact that two or morehistidine residues can cooperate to form very strong metal ioncomplexes. The NTA chelating ligand immobilized on agarose and loadedwith Ni²⁺ has been useful in this method (Hochuli et al., supra; U.S.Pat. No. 5,047,513). It is available commercially through Qiagen, Inc.(Chatsworth, Calif.). However, this resin has the disadvantage that theinterchanges between metal ions and poly-histidine recombinant proteinsare not optimal. Metal leakage can occur, and background proteins cansometimes contaminate purification of recombinant proteins.

A metal chelating gel, i.e., carboxymethylated aspartate (CM-Asp)agarose complexed with calcium, has been used for purifying nativecalcium-binding proteins (Mantovaara, T., H. Pertoft, J. Porath [1989]Biotechnology and Applied Biochemistry 11:564-570; Mantovaara, T., H.Pertoft, J. Porath [1991] Biotechnology and Applied Biochemistry13:315-322; Mantovaara, T., H. Pertoft, J. Porath [1991] Biotechnologyand Applied Biochemistry 13:120-126). However, the Ca²⁺-CM-Asp complexdescribed by Mantovaara et al., has among its disadvantages that it doesnot bind strongly to histidine-tagged recombinant proteins. Anotherdisadvantage, in addition to this inferior binding property, is itsnon-selectivity for histidine tags.

By contrast, the subject invention comprises the CM-Asp chelating ligandcomplexed to a transition metal in an octahedral geometry (coordinationnumber of 6). In this unique configuration, the metal complex can beadvantageously suited for purification of poly-histidine fusedrecombinant proteins. This is a novel use of the CM-Asp ligand and ispart of the subject of the invention herein described.

BRIEF SUMMARY OF THE INVENTION

The present invention concerns a novel IMAC purification method whichemploys immobilized carboxymethylated aspartate (CM-Asp) ligandsspecifically designed for purification of recombinant proteins fusedwith poly-histidine tags. The new purification method is based upon theCM-Asp chelating matrix having the following structure:

A general description of the matrix used in the invention andillustrated above is:

When R₄—R₅—R₆=H:

M=transition metal ion in a 2+ oxidation state with a coordinationnumber of 6;

R₁=a linking arm connecting the nitrogen atom of CM-Asp with R₂;

R₂=a functional linking group through which CM-Asp linking arm R₁ isconnected to R₃;

R₃=a polymer matrix, e.g., those polymer matrices typically used inaffinity or gel chromatography.

When R₁—R₂—R₃=H:

R₄=a linking arm connecting the methylene carbon atom of thecarboxymethyl group of CM-Asp with R₅;

R₅=a functional linking group through which CM-Asp linking arm R4 isconnected to R₆;

R₆=a polymer matrix, e.g., those polymer matrices typically used inaffinity or gel chromatography.

In a preferred embodiment:

M=Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, or Zn²⁺;

R₁=—CH₂CH(OH)CH₂—, —CH₂(OH)CH₂—O—CH₂CH(OH)CH₂—, —(CH₂)₄NHCH₂CH(OH)CH₂—,and —(CH₂)₂NHCH₂CH(OH)CH₂—;

R₂=O, S, or NH; and

R₃=agarose.

In a particularly preferred embodiment:

M=Co²⁺;

R₁=CH₂CH(OH)CH₂;

R₂=O; and

R₃=agarose, cross-linked.

Prior to loading the 6×His recombinant protein to the resin, recombinantcells can be lysed and sonicated. The lysate can then be equilibratedwith an aqueous buffer (pH 8) which itself does not form chelates withthe metal. An example of an aqueous that can be used at this step in thedescribed procedure is 50 mM sodium phosphate (pH 8.0)/10 mM Tris-HCl(pH 8.0)/100 mM NaCl, or the like. The equilibration buffer can containdenaturing agents or detergents, e.g., 10% “TRITON X-100,” 6 Mguanidinium HCl, or the like. After binding the prepared 6×Hisrecombinant protein on the metal CM-Asp chelating resin (the “CM-Aspresin complex”), the protein-bound resin is washed at pH 7.0 or 8.0. Theelution of the protein can be carried out at a constant pH or with adescending pH gradient. In a preferred embodiment, protein elution canbe achieved at a pH of about 6.0 to about 6.3. Alternatively, the 6×Hisrecombinant protein bound to the CM-Asp chelating resin can be washedwith low concentrations (less than 100 mM) of imidazole at pH 8.0 andthen eluted by increasing the imidazole concentration to 40-100 mM.

Also included as an aspect of the subject invention is a scaled-upsynthesis of the CM-Asp derivatized agarose chelating resin. It is animproved version of a previously reported small scale preparation(Mantovaara, T., H. Pertoft, J. Porath [1991] Biotechnology and AppliedBiochemistry 13:315-322). The improvement includes particular conditionsfor oxirane-agarose formation, temperature controlled conjugation ofaspartic acid to the oxirane-agarose, and high ionic strength washing toremove extraneously bound metals. These conditions, temperatures, andionic concentrations are described in detail herein.

An additional application of the subject invention includes screeningfor protein function on a microtiter plate or filter. The additionalapplications for the subject invention also include protein-proteininteraction studies, as well as antibody and antigen purification. Forexample, by immobilization of the Co²⁺ moiety onto 96-well plates byCM-Asp, such plates can be used for quantitation of 6×Histidine-taggedprotein, protein-protein interaction studies, diagnostic screening fordiseases, antibody screening, antagonist and agonist screening fordrugs, and reporter gene assays. Co²⁺ can also be immobilized onto amembrane, e.g., a nylon membrane, by CM-Asp, which can be used to liftproteins from expression libraries to make protein libraries from cells.The membranes also can be used for screening of engineered enzymes.Application of the subject invention can also be extended topurification of any interacting molecule, e.g., nucleic acids or smallco-factors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an outline illustrating a process for purifying recombinant6×His protein using CM-Asp chelating resin.

FIGS. 2A-2B show a comparison of Co²⁺ CM-Asp chelating resin with Ni-NTAon 6×His prepro-α-factor purification under denaturing conditions usingpH gradient.

Legend for FIGS. 2A-2B: lane 1: crude lysate; lane 2: flowthrough; lane3: washed with 6 M Gu-HCl, 0.1 M NaH₂PO₄, pH 8.0; lane 4: washed with 8M urea, 0.1 M NaH₂PO₄; lane 5: washed with 8 M urea, 0.1 M NaH₂PO₄, pH8.0; lane 6: deluted with 8 M urea, 0.1 M NaH₂PO₄, pH 6.3; lane 7:deluted with 8 M urea, 0.1 M NaH₂PO₄, pH 6.3; lane 8: deluted with 8 Murea, 0.1 M NaH₂PO₄, pH 6.3; lane 9: deluted with 8 M urea, 0.1 MNaH₂PO₄, pH 5.9; lane 10: deluted with 8 M urea, 0.1 M NaH₂PO₄, pH 4.5;lane 11: deluted with 6 M Gu-HCl, 0.1 M NaH₂PO₄, 0.2 M acetic acid; laneM: MW size markers.

FIG. 3 shows 6×His tagged DHFR purification by Co²⁺ CM-Asp chelatingresin under native conditions. Legend: Lane 1: clarified lysate; lane 2:flowthrough; lane 3: first wash; lane 4: third wash; lane 5: DHFR finalelution.

FIG. 4 shows 6×His tagged DHFR purification by Co²⁺ CM-Asp chelatingresin under denaturing conditions. Legend: Lane 1: clarified lysate;lane 2: flowthrough; lane 3: first pH 7.0 wash; lane 4: second pH 7.0wash; lane 5: DHFR, first pH 6.0 elution; lane 6: DHFR, second pH 6.0elution.

FIG. 5 shows 6×His tagged DHFR purification by Co²⁺ CM-Asp chelatingresin under native conditions with increasing concentrations ofβ-mercaptoethanol. Legend: lane 1: 20 μl of cell lysate; lanes 2, 4, 6,and 8: 20 μl of flowthrough; lanes 3, 5, 7, and 9: 5 μl of eluant.

FIG. 6 shows yields of 6×His DHFR from cell lysates purified by Co²⁺CM-Asp chelating resin versus Ni-NTA in the presence ofβ-mercaptoethanol. Protein concentrations were determined by Bradfordassay. Yields are expressed as a percentage of total protein in the celllysate.

FIGS. 7A-7B show purification of 6×His GFP by Co²⁺ CM-Asp chelatingresin under native conditions. The GFP bands were detected usingClontech's chemiluminescence Western Exposure Kit and overnight exposureto x-ray film.

Legend: lane 1: clarified lysate; lane 2: flowthrough; lane 3: firstwash; lane 4: first elution; lane 5: second elution; lane 6: thirdelution; lane 7: fourth elution.

FIG. 8 shows biological activity of 6×His GFP purified by Co²⁺ CM-Aspchelating resin. Legend: tube 1: cell lysate; tube 3: flowthrough; tube3: wash; tube 4: first elution; tube 5: second elution; tube 6: thirdelution.

DETAILED DISCLOSURE OF THE INVENTION

The subject method, which employs a CM-Asp metal chelating complex, canadvantageously be used for purification of recombinant proteins having apolyhistidine tail or “tag.”

According to one embodiment of the subject invention, a resin ligand,e.g., CM-Asp, is complexed to a metal other than Ca²⁺, forming aCM-Asp-metal complex. Preferably, the CM-Asp ligand used in the subjectinvention is complexed with one of the transition metals (known as athird-block transition metal), e.g., Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, or Zn²⁺ inan octahedral geometry. Other polymer matrices, e.g., polystyrene (as inmicrotiter plates), nylon (as in nylon filters), SEPHAROSE (Pharmacial,Uppeala, Sweden) or the like, can be used with the subject invention andwould readily be recognized by persons of ordinary skill in the art. Thepoly-histidine tag possesses “neighboring” histidine residues which canadvantageously allow the recombinant protein to bind to these transitionmetals in a cooperative manner to form very strong metal ion complexes.This cooperative binding refers to what is commonly known in the art asa “neighboring histidine effect.” For purposes of the subject invention,and as would be understood by a person of ordinary skill in the art, a“strong” or “very strong” metal ion complex refers to the bond strengthbetween the metal ion and the chelating ligand. A strong or very strongmetal ion complex, for example, allows little or essentially no metalleakage from the complex so that the purified protein, e.g., arecombinant protein having a polyhistidine tag, is not contaminated withundesired or “background” protein from a mixture being purified.

The CM-Asp metal complex offers two available valencies that can formstrong and reversible metal complexes with two adjacent histidineresidues on the surface of the recombinant protein. Another advantage tousing the CM-Asp ligand is its ability to strongly anchor the metal ionwhereby metal ion leaking can be virtually eliminated compared to metalleakage observed for other complex binding agents, e.g., Ni-NTA. In amore preferred embodiment, Co²⁺ can be used as the transition metal withCM-Asp. The Co²⁺-CM-Asp can be less sensitive to reducing agents, suchas β-mercaptoethanol. Metal ion leakage has been shown to remain low,even negligible, in the presence of up to 30 mM β-mercaptoethanol.

One embodiment of the purification process of the subject invention isas follows:

1. Prepare lysate/sonicate containing recombinant 6×His proteinaccording to standard procedures and techniques well known in the art.

2. Bind 6×His protein onto metal-loaded CM-Asp chelating resin atslightly basic pH, e.g., about pH 8.0.

3. Wash protein/resin complex at the same basic pH (about pH 8.0).Optional washes at a pH of about 7.0 or with imidazole additive can alsobe included.

4. Elute pure recombinant 6×His protein with an elution buffer having apH of about 6.0-6.3 or, in the alternative, an elution buffer having apH of about 8.0, plus about 40 to about 100 mM imidazole.

The steps involved in a preferred embodiment of the purification processof the subject invention are illustrated in FIG. 1. The subject processcan be employed batchwise, in spin columns, and in large-scalecontinuous-flow columns.

Buffers used in the above procedures are standard buffers typically usedin similar procedures, with appropriate adjustments and modificationsmade as understood in the art. For example, a high ionic strengthbuffer, e.g., 50 mM phosphate/10 mM Tris/100 mM NaCl can be used, withthe pH adjusted as needed. The phosphate salt component can range from aconcentration of 10-100 mM; Tris from 5-25 mM; and NaCl from 50-200 mM.

Optimal elution conditions depend on the type of impurities, the amountof protein to be purified, and unique properties of the protein, and aredetermined on a case-by-case basis as would be readily recognized by aperson of ordinary skill in the art.

The subject invention also pertains to a method for synthesizingcarboxymethylated aspartate chelating matrices, comprising the steps of:

(a) Michael addition of the α-amino function of monoprotectedα,ω-diamino acids to maleic acid;

(b) deprotecting the ω-amino functionality; and

(c) attaching the chelator primary amine molecule to a solid matrix.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1

Large-Scale Preparation of CM-Asp Chelating Resin

SEPHAROSE CL-6B or CL-6B (Pharmacia, 8.0 L) is washed with ddH₂O,suction dried, and transferred to a 22-L round bottom flask equippedwith mechanical stirring. Epichlorohydrin (about 2.0 L) is added, theSepharose resin mixed to a thick suspension, and allowed to stand atroom temperature for about 20 minutes. A solution of sodium hydroxide(about 560 g) and sodium borohydride (about 48 g) in approximately 6400mL ddH₂O is added and the mixture stirred overnight at ambienttemperature. The oxirane-derivatized resin, collected by filtration, iswashed ten times with ddH₂O (about 10 L each), once with 10% sodiumcarbonate (about 10 L), suction dried, and transferred to a 22 L roundbottom flask. A specimen of the oxirane derivatized resin treated with1.3 M sodium thiosulfate is titrated to approximately pH 7.0 todetermine the oxirane concentration (preferably, >700 μmol/g).

To a solution of sodium hydroxide (approximately 268 g) in about 7.6 LddH₂O is added L-aspartic acid (about 575 g) and sodium carbonate (about1700 g), keeping the temperature below about 25° C. The pH is adjustedto approximately 11.0 and the solution added to the resin. Usingmechanical stirring and a heating mantle, the reaction mixture isbrought to about 80° C. for 4 hours and allowed to cool to roomtemperature overnight. The resin was collected by filtration, washed tentimes with ddH₂O (about 10 L each), once with 10% sodium carbonate(about 10 L), suction dried, and transferred to a 22-L round bottomflask equipped with mechanical stirring.

To an ice-cooled solution of sodium hydroxide (about 900 g) in 12 LddH₂O was added bromoacetic acid (about 3000 g) in approximately 750 gincrements, keeping temperature below about 30° C. Sodium carbonate(about 660 g) is added and the pH is adjusted to about 10. The resin isreacted with the solution at ambient temperature overnight. The resin iscollected by filtration, washed six times with ddH₂O (about 10 L each),six times with 10% acetic acid, and ten times with ddH₂O. Washing iscontinued with ddH₂O until the pH reached about 6.0 by litmus paper. TheCM-Asp chelating resin was suction dried in preparation for metalloading.

EXAMPLE 2

Preparation of C-Linked CM-Asp Chelating Resin

N⁶-Carbobenzyloxy-L-lysine (6.15 g) and excess maleic acid (17.6 g) weredissolved in 2 M NaOH (35 mL). The solution was refluxed for 24 hr andallowed to cool to ambient. The pH was adjusted to 3 with 6 M HCl andchilled. The mixture was filtered to remove the unreacted maleic acidwhich was washed with water (15 mL). The filtrate and washings werecombined and evaporated. The residue was dissolved in 4% ammoniumformate (120 mL) and degassed briefly. 10% Pd/C (600 mg) was added andthe mixture was refluxed under Ar with stirring for 5 hr. The mixturewas filtered through celite and the filtrate evaporated. The residue wasdissolved in a solution of sodium hydroxide (1.3 g) and sodium carbonate(8.7 g) in dd H₂O (40 mL). The final pH was adjusted to 11. Thissolution is added to the oxirane-derivatized resin (40 mL bed volume)prepared as described in Example 1. The mixture was stirred at 60° withmechanical stirring for 4 hr and at ambient for 16 hr. The resin wascollected by filtration, washed with ddH₂O (6×100 mL), 10% HOAc (6×100mL), and ddH₂O (6×100 mL) or until the pH reached about 6 by litmuspaper test. The C-Linked CM-Asp chelating resin was suction dried inpreparation for metal loading.

EXAMPLE 3

Metal Loading of CM-Asp Chelating Resin

The CM-Asp chelating resin of Example 1 (about 1 L of suction dried bedvolume) is treated with a transition metal ion solution, e.g., 2 L ofeither 200 mM of cobalt chloride hexahydrate, nickel sulfatehexahydrate, copper sulfate pentahydrate, or zinc chloride, according tothe metal ion deserved. The resin is reacted with the 200 mM metalsolution at room temperature for approximately 72 hours and thencollected by filtration. The metal loaded CM-Asp chelating resin iswashed five times with ddH₂O (about 1 L each), two times with 100 mMNaCl (about 1 L each), six times with ddH₂O (about 1 L each), and oncewith 20% aq. ethanol (about 1 L). The resin can be stored in 20% aq.ethanol.

EXAMPLE 4

Comparison of Co²⁺ CM-Asp Resin with Ni²⁺-NTA on Recombinant 6×HisPrepro-α-Factor Under Denaturing Conditions Using pH Gradient

For a qualitative comparison of the purification of Co²⁺ CM-Aspchelating resin and Ni-NTA under denaturing conditions, the C-terminal,6×His-tagged prepro-α-factor of S. cerevisiae was expressed in E. coli.One gram bacterial cell pellet was lysed in 6 M guanidinium-HCl (Gu-HCl)and 0.1 M NaH₂PO₄, pH 8.0. Three milliliters of clarified lysate wasloaded onto a Co²⁺ CM-Asp chelating resin gravity flow column. Theresin-proteins mixture was washed with 8 M urea, 0.1 M NaH₂PO₄, pH 8.0,and deluted with 8 M urea, 0.1 M NaH₂PO₄ at three different pHs, 6.3,5.9, and 4.5. Finally, all bound proteins were deluted with 6 M Gu-HCl,0.1 M NaH₂PO₄, 0.2 M acetic acid. Samples from each step were loaded ona 12% polyacrylamide/SDS gel, electrophoresed, and the gel was stainedwith Coomassie blue. The 6×His-tagged prepro-α-factor was deluted at pH6.3 as a single prominent band on the gel.

In the same manner, 3 ml of clarified lysate was loaded onto a Ni-NTAgravity flow column. The resin-proteins mixture was washed and delutedthe same as above. Samples from each step were loaded on a 12%polyacrylamide/SDS gel, electrophoresed, and the gel was stained withCoomassie blue. There were more than 10 protein bands in elution at pH6.3. The 6×His-tagged prepro-α-factor was a minor band among them. Themajority of the protein was deluted at pH 4.5 without any othercontaminant proteins. This demonstrated that the highly purified6×His-tagged prepro-α-factor was deluted from Co²⁺ CM-Asp chelatingresin at the conditions (pH 6.3) under which Ni-NTA was still releasingcontaminants. The affinity of Co²⁺ CM-Asp chelating resin to6×His-tagged prepro-α-factor was more selective than Ni-NTA to theprotein.

Results show that highly purified 6×His-tagged protein elutes from Co²⁺CM-Asp chelating resin while Ni-NTA is still releasing contaminants. SeeFIGS. 2A-2B: FIG. 2A: Results after using 1 ml of Co²⁺ CM-Asp chelatingresin. FIG. 2B: Results after using 1 ml of nickel-NTA.

EXAMPLE 5

Recombinant 6×His DHFR Purification with CM-Asp Resin Under NativeConditions

N-terminal, 6×His-tagged mouse dihydrofolate reductase (DHFR, MW 20.3kDa) was expressed in E. coli cells. Cells were then pretreated with0.75 mg/ml lysozyme and disrupted in lysis buffer (100 mM NaH₂PO₄, 10 mMTris-HCl, pH 8.0) by mechanical shearing, 800 μl of the clarified lysatewas applied to 100 μl of Co²⁺ CM-Asp chelating resin, pre-equilibratedwith lysis buffer, and washed with one ml of lysis buffer three times.All bound protein was deluted by 300 μl of 100 mM EDTA, pH 8.0. Twentymicroliters of lysate and 40 μl of each subsequent fraction from elutionwere run on a 12% polyacrylamide/SDS gel. The gel was stained withCoomassie blue. One single protein band was shown at a position of MW20.3 kDa. Results showed the selective binding affinity of Co²⁺ CM-Aspchelating resin to 6×His-tagged DHFR under native purificationconditions. No discernable binding of host proteins occurred.

Results show that Co²⁺ CM-Asp chelating resin has selective bindingaffinity for 6×Histidines. No discernable binding of host proteinsoccurred. See FIG. 3.

EXAMPLE 6

Recombinant 6×His DHFR Purification with CM-Asp Resin Under DenaturingConditions

N-terminal 6×His-tagged mouse DHFR was expressed in a 25-ml culture ofE. coli. Cells were pelleted, resuspended in lysis buffer (100 mMNaH₂PO₄, 10 mM Tris-HCl, 8 M urea, pH 8.0), and disrupted by vortexing.Six hundred microliters of clarified lysate were applied to a Co²⁺CM-Asp chelating resin spin column containing 0.5 ml of Co²⁺ CM-Aspchelating resin-NX metal affinity resin and centrifuged for 2 minutes at2,000×g. The column was washed twice with 1 ml of wash buffer (100 mMNaH₂PO₄, 10 mM PIPES, pH 7.0), and bound proteins were deluted with 600μl of elution buffer (20 mM PIPES, 100 mM NaCl, 8 M urea, pH 6.0).Twenty microliters of lysate and 40 μl of each subsequent fraction fromthe elution were loaded onto a 12% polyacrylamide/SDS gel andelectrophoresed. The gel was stained with Coomassie blue. One singleprotein band was shown at the position of 20.3 kDa. Results showed theselective binding affinity of Co²⁺ CM-Asp chelating resin to6×His-tagged DHFR under denaturing conditions. The binding properties ofCo²⁺ CM-Asp chelating resin to 6×histidines allow proteins deluted undermild pH conditions (pH 6.0) that protect protein integrity.

Results show that bound protein can be deluted at mild pH (pH 6.0). Thisindicates that the binding properties of Co²⁺ CM-Asp chelating resinallow protein elution under mild pH conditions that protect proteinintegrity. See FIG. 4.

EXAMPLE 7

Recombinant 6×His DHFR Purification with CM-Asp Resin Under NativeConditions with Increasing Concentrations of Beta-Mercaptoethanol

N-terminal, 6×His-tagged mouse DHFR was expressed in E. coli.Twenty-five milliliters of cell culture were disrupted in 2 ml ofsonication buffer (100 mM NaH₂PO₄, 10 mM Tris-HCl, and 100 mM NaCl, pH8.0) by freezing and thawing. Then, 2.66 ml of clarified lysate wereapplied to a 200-μl Co²⁺ CM-Asp chelating resin gravity flow column,pre-equilibrated with the sonication buffer. The proteins/resin mixtureswere washed three times with sonication buffer, pH 8.0. All boundproteins were deluted with 600 μl of 100 mM EDTA, pH 8.0. To test theeffect of β-mercaptoethanol on the Co²⁺ CM-Asp chelating resinpurification under native conditions, all buffers used here containedeither 0, 10, 20, or 30 mM β-mercaptoethanol. Samples from each elutionwere electrophoresed on a 12% polyacrylamide/SDS gel, and the gel wasstained with Coomassie blue. One single protein band at the position ofMW 20.3 kDa was shown from all elutions. The presence ofβ-mercaptoethanol did not obsolete the purity of 6×His-tagged DHFRpurified by Co²⁺ CM-Asp chelating resin. With up to 30 mMβ-mercaptoethanol in all purification buffers, there was no predominantband at 20.3 kDa in flowthroughs, indicating that no loss of metaloccurred during protein purification using Co²⁺ CM-Asp chelating resinin the presence of β-mercaptoethanol.

Results show that with up to 30 mM β-mercaptoethanol in the purificationbuffer, there is no predominant band at 20.3 kDa in the flowthrough,indicating that no loss of metal occurred during protein purificationusing Co²⁺ CM-Asp chelating resin in the presence of β-mercaptoethanol.See FIG. 5.

EXAMPLE 8

Yields of 6×His DHFR from Cell Lysates Purified by CM-Asp Versus Ni-NTAin the Presence of Beta-Mercaptoethanol

N-terminal, 6×His-tagged DHFR was expressed and purified by Co²⁺ CM-Aspchelating resin under native conditions as described in Example 7.Protein concentrations were determined by Bradford assay. Yields wereexpressed as a percentage of total protein in the cell lysate. Theyields of purified 6×His-tagged DHFR were 14%, 28%, 34%, and 35%respectively, with β-mercaptoethanol present in purification buffers atthe concentrations of 0, 10, 20, and 30 mM. The protein was purified byNi-NTA under the same native conditions; the yields of purified6×His-tagged DHFR were 4%, 8.8%, 3.4%, and 4% respectively withβ-mercaptoethanol present in purification buffers at the concentrationsof 0, 10, 20, and 30 mM. The yields of purified 6×His-tagged DHFR weresignificantly higher when using Co²⁺ CM-Asp chelating resin compared toNi-NTA under native purification conditions with β-mercaptoethanol. Thisindicates that the metal ion on Co²⁺ CM-Asp chelating resin is stronglyanchored to SEPHAROSE beads by a CM-ASP metal chelator that is ideal forbinding octahedral metals.

Results show that the yields of purified 6×His DHFR are significantlyhigher at 10, 20, and 30 mM β-mercaptoethanol using Co²⁺ CM-Aspchelating resin compared to using Ni-NTA. This indicates that the metalion on Co²⁺ CM-Asp chelating resin is strongly anchored to sepharosebeads by CM-Asp metal chelator that is advantageous for bindingoctahedral metals. See FIG. 6.

EXAMPLE 9

Purification of 6×His GFP by Co²⁺-CM-Asp Under Native Conditions

N-terminal, 6×His-tagged green fluorescent protein (GFP) was expressedin E. coli cells. Cells were pelleted, resuspended in sonication buffer(100 mM NaH₂PO₄, 10 mM Tris-HCl, and 100 mM NaCl, pH 8.0), and disruptedby freezing and thawing three times. Two milliliters of clarified lysatewere applied to 400 μl of Co²⁺ CM-Asp chelating resin, pre-equilibratedwith sonication buffer, and washed three times with 2 ml of sonicationbuffer, pH 8.0. The 6×His-tagged GFP was deluted with 400 μl of 75 mMimidazole buffer containing 20 mM Tris-HCl and 100 mM NaCl, pH 8.0.Samples from each purification step were loaded onto a 12%polyacrylamide/SDS gel, electrophoresed, and the gel was stained withCoomassie blue. One single band was shown at the position of MW 27.8 kDain the elution with 75 mM imidazole. This demonstrated that 6×His-taggedGFP selectively bound on Co²⁺ CM-Asp chelating resin and can be delutedwith low concentration of imidazole under native purificationconditions.

Samples from each purification step were also loaded on a 12%polyacrylamide/SDS gel, electrophoresed, and transblotted to a PVDFmembrane. The proteins on the blot were probed with anti-GFP monoclonalantibody. One single GFP band was clearly shown in the samples of celllysate and elution. There was no GFP band shown in flowthrough, whichindicated that all expressed GFP in cell lysate was bound to Co²⁺ CM-Aspchelating resin.

Results show that (FIG. 7A) Coomassie blue stained gel shows one singleband in 75 mM imidazole elution. This indicates that 6×Histidinesselectively bound on Co²⁺ CM-Asp chelating resin. Western analysis datashows no GFP in flowthrough which indicates the high affinity betweenCo²⁺ CM-Asp chelating resin and 6×histidines (FIG. 7B).

EXAMPLE 10

Biological Activity of 6×His GFP Purified by Co²⁺ CM-Asp

N-terminal, 6×His-tagged GFP was expressed in E. coli. Cell lysate wasprepared as described in Example 7. The cell lysate was loaded onto a2-ml Co²⁺ CM-Asp chelating resin Disposable Gravity Column, and purifiedusing the Batch/Gravity Flow column purification method as described inExample 9. The column was washed with sonication buffer three times anddeluted with 100 mM EDTA, pH 8.0. Samples were collected in microfugetubes from each purification step. The fluorescence of all collectedsamples was detected using an UltraLum Electronic U.V. Transilluminator.Samples of cell lysate and elution showed strong fluorescence. Thisexperiment demonstrated that 6×His-tagged GFP can be purified tohomogeneity by Co²⁺ CM-Asp chelating resin under native conditions andmaintains biological activity.

The photo of samples from each purification step shows that GFP can bepurified to homogeneity by Co²⁺ CM-Asp chelating resin under nativeconditions, and the fluorescence indicates that GFP purified by Co²⁺CM-Asp chelating resin still maintains its biological activity. See FIG.8.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

What is claimed is:
 1. An immobilized metal ion affinity chromatographypurification method for purification of recombinant proteins, saidmethod comprising: a) providing carboxymethylated aspartate ligandcomplexed with a transition metal ion in a 2+ oxidation state, having acoordination number of 6; b) loading a mixture of cell lysate comprisinga recombinant protein having a polyhistidine tail to bind with saidligand; and c) eluting said recombinant protein with a suitable elutantto obtain a purified recombinant protein, wherein said transitionmetal-complexed carboxymethylated aspartate ligand forms acarboxymethylated aspartate chelating matrix which comprises saidtransition metal and a polymer matrix, wherein said carboxymethylatedaspartate chelating matrix has the structure

wherein R1—R2—R3=H; M=transition metal ion in a 2+ oxidation state witha coordination number of 6; R4=a linking arm connecting the methylenecarbon atom of the carboxymethyl group of Cm-Asp with R5; R5=afunctional linking group through with CM-Asp linking arm R4 is connectedto R6; and R6=a polymer matrix.
 2. An immobilized metal ion affinitychromatography complex comprising a carboxymethylated aspartate ligandand a transition metal chelated therto, wherein said transition metalion has a 2+ oxidation state and a coordination number of 6, whereinsaid complex has the structure:

wherein R1—R2—R3=H; M=transition metal ion in a 2+ oxidation state witha coordination number of 6; R4=a linking arm connecting the methylenecarbon atom of the carboxymethyl group of Cm-Asp with R5; R5=afunctional linking group through with CM-Asp linking arm R4 is connectedto R6; and R6=a polymer matrix.
 3. The complex according to claim 2,wherein said polymer matrix comprises a polymer matrix suitable for usein affinity or gel chromatography.
 4. The complex according to claim 2,wherein M=Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, or Zn²⁺; R₄=—(CH₂)₄NHCH₂CH(OH)CH₂— or—(CH₂)₄NH—; R₅=O, S, NH, or CO; and R₆=agarose or polystyrene.
 5. Thecomplex, according to claim 4, wherein M=Co²⁺; R4=—(CH₂)₄NHCH₂CH(OH)CH₂—or —(CH₂)₄NH—; R5=O or CO; and R6=agarose, cross linked agarose, orpolystyrene.